Streptococcus gordonii is an initial colonizer of the biofilm that forms on the saliva-coated tooth surface. These pioneer colonizers and their extracellular products form a substratum with host oral components to which subsequent colonizing bacteria can attach. Accordingly, S. gordonii plays a pivotal role in the establishment of the mature oral biofilm through mechanisms such as host and bacterial adhesin-receptor, metabolic, and intercellular signaling interactions. The establishment and maintenance of a healthy oral microbiome requires a favorable balance of these ecological interactions. The working hypothesis of the proposed studies is that identification of S. gordonii genes essential for species survival and proliferation under synergistic or antagonistic conditions will provide essential insights into community dynamics that favor or disfavor a state of oral health. The goal of this exploratory project is to use a genome-wide approach to identify and examine genes involved in S. gordonii fitness under selected in vitro model conditions relevant to those in the oral cavity. We propose a state-of-the-art, Tn-Seq approach to construct a saturated library of transposon genome insertions in S. gordonii strain Challis, a widely-used model of commensal oral streptococci. The usefulness of the library will be confirmed by growth under test conditions that will allow identification of mutants with insertions in genes that are essential for growth and fitness. Mutants with disruptions of genes conferring fitness will decrease in frequency in the population during the course of the experiment; in contrast, mutants in the library with insertions in nonessential genes will be maintained at an equal or increased frequency. Recovered DNA from the entire population will be collected at the end of experiment and compared to the input library via a high throughput sequencing of the chromosomal DNA flanking the integrated transposon. Interrupted and hence, less or non- functional genes will be identified by comparison of the transposon-flanking nucleotide sequence to the reference S. gordonii genome sequence. The frequency of recovery of each mutant will be used to calculate the fitness of each gene under the experimental conditions. Conditions to be studied will include biofilm formation and planktonic conditions important for colonization in presence of whole human saliva, serum, and dual species cultures with Actinomyces oris and Filifactor alocis, representing bacterial species that have been shown to promote or impede, respectively, S. gordonii colonization in biofilms. The roles of selected genes identified as playing potential roles in fitness will be further examined by construction of specific mutant strains by allelic exchange and their complements to verify their roles in the survival and proliferation of S. gordonii during biofilm development. These studies will provide an important foundation for future bacterial interaction and survival studies. Understanding the roles of genes that favor the commensal oral microbiota will provide essential insights into preventing the microbial dysbiosis that can lead to oral disease.